Vascular SMCs are arranged circumferentially in arteries in multiple layers, either embedded between layers of elastin lamellae in large elastic arteries or in a matrix of connective tissue in smaller muscular arteries. For contractile function, SMCs express smooth muscle specific isoforms of o-actin and (3-myosin, which multimerize to form thin and thick filaments, respectively. We have determined that mutations in the genes encoding SMC a-actin and p-myosin, ACTA2 and MYH11, along with the kinase that controls SMC contraction, myosin light chain kinase (MYLK), predispose individuals to vascular diseases, including thoracic aortic aneurysms and aortic dissections and occlusive vascular diseases, such as early onset coronary artery disease and stroke. The Program Project Grant (PPG) will test the hypothesis that the ACTA2, MYH11 and MYLK mutations lead to thoracic aortic and disease and/or occlusive vascular diseases due to a differential SMC response to biomechanical stresses resulting from dysfunction of the contractile unit. We hypothesize that mutations In these genes cause a loss of function, specifically loss of regulation or altered force output of contractile unit in SMCs in ascending aorta, resulting in increased biomechanical stresses and activation of SMC pathways leading to thoracic aortic aneurysms and aortic dissections. In contrast, we hypothesize that the occlusive vascular diseases associated with a subset of ACTA2 mutations, and possibly MYH11 variants, result from a SMC gain of function in muscular arteries, specifically increased SMC proliferation as a consequence of altered focal adhesions and activation of PDGFR-P receptors in response to abnormal cellular force generation. To test these hypotheses. Project 1 and 2 will assess the Impact of ACTA2 and MYH11 genetic variants on kinetics, motility, regulation, and filament formation in vitro. Project 3 will assess age-dependent derangements in specific signaling modules responsible for vasomotor responsiveness in vascular SMCs in mouse models of select ACTA2, MYH11 and MYLK mutations. Project 4 will Investigate SMC cellular pathways leading to thoracic aortic disease and SMC proliferation in these same mouse models. RELEVANCE (See instructions): The proposed studies will provide insight into mechanisms leading to the vascular diseases due to disruption of contraction of the muscle cells that surround the arteries. These data may provide the basis for targeted therapeutics to prevent these vascular diseases, including thoracic aortic aneurysms and dissections, strokes and coronary artery disease.